Individual variation in inflammatory responses regulates onset and severity of multiple sclerosis (MS) and other types of brain injury. Initiation, amplification, and resolution of these inflammatory responses occur in part through innate immune signaling mediated by macrophages and related immune cells. This laboratory has discovered novel innate immune signaling pathways in human macrophages that are regulated by intracellular splice variants of voltage-gated sodium channels. These channels regulate pattern recognition of dsRNA, intracellular signaling, vesicular trafficking, and transcription of anti-viral genes. In a mouse model of MS, expression of one of these channels, human macrophage SCN5A, in mouse macrophages reduced disease severity and enhanced tissue repair. Recently published work demonstrates that a newly discovered channel variant, human macrophage SCN10A, acts in a synergistic manner with SCN5A to regulate RNA processing of a transcript that encodes a DNA repair protein, PPP1R10. New preliminary data demonstrate individual variation in regulation of PPP1R10 expression. New data also reveal that SCN10A localizes to mitochondria during cellular injury and regulates ATP production. The objective of this revised proposal is to characterize these innate immune signaling mechanisms in human cells and an animal model. The central hypothesis is that human macrophage SCN10A and SCN5A prevent cell and tissue injury through enhancement of DNA repair and maintenance of cellular bioenergetics. This hypothesis will be assessed in three aims: 1) Analyze how human macrophage channel variants regulate PPP1R10 protein expression, 2) Determine how the human macrophage channel variants regulate mitochondrial function, and 3) Characterize how macrophage SN10A and SCN5A prevent tissue injury. For Aim 1, the proposed model is that endogenous signals of cellular injury activate human macrophage SCN10A and SCN5A to initiate a calcium-dependent nuclear signaling pathway that regulates expression of the DNA repair protein PPP1R10. It is hypothesized that individual variation in this pathway increases the risk of tissue injury in inflammatory diseases such as MS. For Aim 2, it is proposed that human macrophage SCN10A localizes to mitochondria during cellular injury to transiently increase mitochondrial ATP production. It is also hypothesized that SCN10A and SCN5A regulate mitophagy, a cellular protective mechanism. For Aim 3, it is postulated that macrophages that express human variants of SCN5A and SCN10A prevent tissue injury in inflammatory lesions through enhancement of DNA repair and maintenance of bioenergetics. These hypotheses will be tested using multidisciplinary approaches in primary cultures of human macrophages; macrophages, microglia, and neurons derived from human induced- pluripotent stem cells; and in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. The expectations are that we will identify novel regulatory mechanisms of bioenergetics and tissue repair that are relevant to MS and related diseases. The long-term goals are to develop new biomarkers of disease susceptibility and severity and identify novel therapeutic strategies that prevent and reduce long- term disability of Veterans with MS.